This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding protein kinase enzymes in plants and seeds.
Calcium can function as a signal which activates gene expression via stimulation of calcium-dependent protein kinases. Calcium-dependent protein kinases (CDPKs) represent a family of protein kinases which are proposed to contain, in a single polypeptide, both a kinase domain and an adjoining calmodulin-like domain with four calcium-binding motifs (Harper, J. F., et al. (1991) Science 252:951-954). Some CDPK proteins kinase have been isolated that require calcium but not calmodulin for activity. Research has shown that multiple CDPK isoforms are present in Arabidopsis thaliana and other plants and that plant CDPKs may play a pivotal role in the regulation of many cellular process such as stress response (WO 98/26045) and male gametophyte formation (WO 97/35968).
Glycogen synthase kinase-3 (GSK-3) in animal cells is a serine/threonine kinase protein, which is involved in the regulating the activity of several transcription factors including the DNA-binding activity of the c-jun/AP1 transcription factor. AP1 is a transcription factor that recognizes a specific enhancer target DNA sequence and when bound to enhancer regions stimulates promoter transcriptional activity. AP1 is composed of several polypeptides of which c-jun is the major component. Several plant GSK-3 proteins have been identified that have homology to GSK-3 gene family of protein kinases (Bianchi et al. (1994) Mol. Gen. Genet. 242(3):337-345).
There is a great deal of interest in identifying the genes that encode protein kinase enzymes involved in the control of gene expression in plants. These genes may be used to modulate or control protein expression in plant cells. Accordingly, the availability of nucleic acid sequences encoding all or a portion of a regulatory protein kinase would facilitate studies to better understand gene regulation in plants, and provide genetic tools to permit more accurate control and manipulation of gene expression in plants.
The instant invention relates to isolated nucleic acid fragments encoding protein kinase enzymes. Specifically, this invention concerns an isolated nucleic acid fragment encoding a calcium dependent phosphorylase kinase or glycogen synthase kinase-3 and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding a calcium dependent phosphorylase kinase or glycogen synthase kinase-3. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding calcium dependent phosphorylase kinase or glycogen synthase kinase-3.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a protein kinase selected from the group consisting of calcium dependent phosphorylase kinase and glycogen synthase kinase-3.
In another embodiment, the instant invention relates to a chimeric gene encoding a calcium dependent phosphorylase kinase or glycogen synthase kinase-3, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a calcium dependent phosphorylase kinase or glycogen synthase kinase-3, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a calcium dependent phosphorylase kinase or glycogen synthase kinase-3, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of a calcium dependent phosphorylase kinase or glycogen synthase kinase-3 in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a calcium dependent phosphorylase kinase or glycogen synthase kinase-3; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of calcium dependent phosphorylase kinase or glycogen synthase kinase-3 in the transformed host cell.
An additional embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a calcium dependent phosphorylase kinase or glycogen synthase kinase-3.